1. Immunohistochemical Diagnosis of Mouse Hepatitis
Virus and Mycoplasma pulmonis Infection
with Murine Antiserum
C. T. Liang*,†, S. C. Wu*, Y. T. Huang*, Y. C. Lin*, W. J. Chang*,
J. Y. Chou‡, S. C. Liang*,‡ and C. H. Liu†
*National Laboratory Animal Center, National Applied Research Laboratories, Nan-Kang, Taipei 115, †
Department and
Graduate Institute of Veterinary Medicine, College of Bioresources and Agriculture, National Taiwan University,
Taipei 106, and ‡
Laboratory Animal Center, National Defense Medical Center, Taipei, Taiwan, ROC
Summary
This study established a modified alkaline phosphatase-labelled avidin-biotin-complex (ABC-AP) method
for diagnosis of mouse hepatitis virus (MHV) and Mycoplasma pulmonis infection from formalin-fixed,
paraffin wax-embedded sections, murine antibody-positive serum being used as the primary reagent. With
this method, MHV antigen in cAnNCrj.Cg-Foxn1nu
/Foxn1nu
mice and M. pulmonis antigen in Wistar rats
were immunolabelled in tissue sections. MHV antigen was clearly detected in samples of liver, stomach,
caecal and colonic mucosa, and spleen. M. pulmonis antigen was demonstrated on the luminal surface of
bronchiolar epithelial cells. This method may prove useful in diagnosis when commercial antisera are
unavailable or when immunosuppression prevents serological diagnosis.
q 2004 Elsevier Ltd. All rights reserved.
Keywords: bacterial infection; mouse; mouse hepatitis virus; Mycoplasma pulmonis; viral infection
Introduction
Avidin-biotin-complex immunohistochemistry
offers a sensitive, reliable method for the detection
of pathogens in tissues. Mouse hepatitis virus
(MHV) and Mycoplasma pulmonis are the most
prevalent pathogens of laboratory mice and rats
(National Research Council, 1991). Respiratory
strains of MHV infect the nasal mucosa and then
spread to the liver, lymphoid tissue, uterus,
placenta, peritoneum, brain, vascular endothelium
and bone marrow by the lymphatic or vascular
route, or directly via olfactory pathways from the
nose to the brain. Enterotropic strains of MHV
usually infect the intestinal mucosal epithelium
and nasal passages, with less involvement of
other tissues (National Research Council, 1991;
Compton et al., 1993; Liang et al., 1995).
M. pulmonis, an extracellular pathogen of mice
and rats, preferentially colonizes the luminal sur-
face of respiratory epithelium, the middle ear and
endometrium (National Research Council, 1991;
Percy and Barthold, 1993). Infection is usually
diagnosed by microbial isolation, serological test-
ing and histological examination. Microbial iso-
lation requires multisite culture for reliable results,
and serological methods are hampered by cross-
reactivity between different species of mycoplasma
(Cassell et al., 1981). Serological testing is useful
during the active and convalescent phases of
disease (Feldman, 2001), but screening of immu-
nodeficient mice is unreliable (Casebolt et al.,
1997). Immunohistochemistry is widely used to
J. Comp. Path. 2004, Vol. 131, 214–220
www.elsevier.com/locate/jcpa
0021–9975/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi: 10.1016/j.jcpa.2004.04.003
Correspondence to: C. H. Liu, Department of Veterinary Medicine,
National Taiwan University, No. 1, Section 4, Roosevelt Road, Taipei
106, Taiwan.

2. detect microbial antigen in tissue sections from
naturally infected animals, except when specific
primary antibodies are commercially unavailable or
excessively expensive. In such cases, the use of
positive antiserum from infected members of the
homologous species is of potential valve. Although
the use of homologous primary antiserum is not
usually applicable in immunohistochemistry, a
suitable method was recently described by Lu and
Partridge (1998). In the present study, an avidin-
biotin-complex and alkaline phosphatase (ABC-AP)
method was developed in which ELISA-positive
murine antiserum was used as the primary antibody
for the diagnosis of MHVand M. pulmonis infection.
Materials and Methods
Animals
Experiment 1. Specific-pathogen-free cAnNCrj.Cg-
Foxn1nu
/Foxn1nu
and Foxn1nu/þ
nude mice, orig-
inally obtained from Charles River Laboratories
(Yokohama, Kanagawa, Japan), were maintained at
the National Laboratory Animal Center (NLAC) in
Taiwan. Thirty of these mice, consisting of 20
homozygous males aged 5 weeks and 10 homo-
zygous females aged 9 weeks were sold, to a unit
that maintains animals for use in research in Taipei
City, on February 2 and March 8, 2000, respectively.
Animals were housed in sterile microisolator cages
(Laboratory Products, Maywood, NJ, USA), kept in
animal cabinets (Nu-air; Plymouth, MN, USA), and
placed in conventional rooms. Sterile water and
commercial rodent chow (PMI Feeds, St Louis,
MO, USA) was provided ad libitum. Bedding
changes, water replenishment and supply of food
were carried out in a class-II biological safety
cabinet. The animal houses were maintained at
20 to 25 8C with a 12-h light/dark cycle.
At 42–49 days after arrival at the unit, the male
nude mice had become emaciated, anorexic and
dehydrated, with scaly skin, hunched posture,
diarrhoea and ocular discharge, and 50% had
died. The female nude mice showed similar but less
severe signs. Four male and two female nude mice
were humanely killed at the age of 15–16 weeks
and samples were taken back to the NLAC for
pathological examination.
Experiment 2. Sixty 6-week-old female Wistar rats
from the NLAC were transferred to a clean
conventional local area and housed in autoclaved
microisolators. Bedding was changed twice a week
in class II biohazard cabinets. Animals were main-
tained at a room temperature of 20–25 8C and
humidity of 50–70%, with a 12-h light /dark cycle.
Sterile water and commercial rodent chow (PMI
Feeds) were provided ad libitum. Bedding (Beta-
chip; Northeastern Inc., Warrensburg, NY, USA),
polysulfone cages (Laboratory Products) and
supplies were all autoclaved. A proportion (30%)
of the animals showed sneezing 5–6 weeks after
arrival. Thirteen of the affected animals, now aged
12–24 weeks, were killed for diagnostic evaluation.
Necropsy revealed consolidation of the apical and
cardiac lobes of the lungs. ELISA screening for
pneumonia virus of mice (PVM), Sendai virus,
lymphocytic choriomeningitis virus (LCMV), and
sialodacryoadenitis virus (SDAV) was negative. Anti-
body titres of three rats for M. pulmonis, assessed by
ELISA score, were 6.07 to 17.22. ELISA results were
interpreted on the basis of the method described
below.
Preparation of ELISA-positive Sera
The ELISA monitoring programme and diagnostic
service of the NLAC were used to detect the
following infectious agents: pneumonia virus of
mice (PVM), reovirus (Reo-3), Sendai virus, lym-
phocytic choriomeningitis virus (LCMV), Theiler’s
murine encephalomyelitis virus (GD VII), minute
virus of mice (MVM), mouse hepatitis virus (MHV),
mouse adenovirus (Mad), ectromelia virus,
Kilham’s rat virus (KRV), sialodacryoadenitis virus
(SDAV/RCV), M. pulmonis, hantavirus, K virus, and
Clostridium piliforme. The procedure followed the
Charles River ELISA scoring system (Serology
Method Manual; Charles River Laboratories,
Wilmington, MA, USA). Briefly, 50 ml of serum
sample, diluted 1 in 60 in BLOTTO (Bovine Lacto
Transfer Technique Optimizer; 5% non-fat dry milk
in phosphate-buffered saline (PBS)) (Johnson et al.,
1984), were added to each appropriate antigen well
and control well. The plate was covered and
incubated for 40 min at 37 8C. After several wash-
ings, 50 ml of horseradish peroxidase-conjugated,
affinity-purified horse anti-mouse or anti-rat IgG
(Kirkegaard and Perry Laboratories, Maryland,
USA), depending on species, were added to each
well. After incubation for 40 min at 37 8C, the plate
was washed again. One hundred ml of 0.4 mM
ABTS-2.0 mM H2O2 chromogenic substrate were
then added to each well and the plate was
incubated at room temperature for 40 min. Absor-
bance was determined colorimetrically at 405 nm
with an ELISA microplate reader (Thermo-max;
Molecular Devices, Sunnyvale, CA, USA). Absor-
bance values were transmitted from the ELISA
reader to a personal computer (PC) where
they were converted to scores by dividing by 0.13.
Diagnosis of Laboratory Animal Infections 215

3. The denominator of 0.13 divided net absorbance
values of 0.13 to 1.3 into scores of 1 to 10. Integer
scores were read and interpreted by comparison
with the 3-decimal absorbance values. The PC was
also used to compute net scores (Scoreantigen minus
Scoretissue control). A result was considered
non-specific and recorded as tissue control (TC)
when both Scoreantigen and Scoretissue control were 2
or above. Provided that the Score tissue control was
lower than 2 (absorbance values lower than 0.26),
net scores were interpreted as follows: 0–1, nega-
tive; 2–3, borderline; $3, positive. When the test
serum was interpreted as “single agent (i.e., MHV)-
positive”, and the net score was $10, serum samples
were collected and stored at 220 8C until used as
primary antibody for immunohistochemistry.
Pathological Examination
The lungs, trachea, lymph nodes, heart, liver,
spleen, small intestine, stomach, kidneys, urinary
bladder, adrenal glands, skin and brain from
affected animals in experiments 1 and 2 were
fixed in 10% neutral buffered formalin. The tissue
samples were processed by routine methods to
paraffin wax-embedded blocks. Sections (6 mm)
were stained with haematoxylin and eosin (HE).
Immunohistochemistry
The Mouse on Mouse kit (M.O.M.e; Vector
Laboratories, Burlingame, CA, USA) was used for
the immunohistochemistry study of MHV infection
in experiment 1. The primary antibodies were
ELISA-positive, mouse anti-MHV sera (ELISA
score; 11.2–14.4). Infection with PVM, Reo-3,
Sendai virus, LCMV, GD VII, MVM, Mad, ectrome-
lia virus, K virus, M. pulmonis and polyoma virus
were ruled out on the basis of ELISA results.
Tissue sections were dewaxed in xylene and
rehydrated in a graded alcohol series. Antigen
unmasking was performed by immersion of sec-
tions in Vector antigen unmasking solution 1% in
PBS and boiling for 5 min in a 1450-W microwave
oven (RE-C102; Sampo Co., Taiwan). The sections
were then immersed in cool PBS for 10 min, rinsed
in PBS, and incubated with trypsin (Sigma Chemi-
cal Co., St Louis, MO, USA) 0.1% in PBS for 5 min
at 40 8C. Endogenous peroxidase activity was
quenched with hydrogen peroxide 0.3% in metha-
nol for 5 min at 40 8C. The sections were then
rinsed in PBS, incubated for 30 min at 40 8C in
mouse M.O.M.e IgG blocking reagent, rinsed with
PBS, and incubated in M.O.M.e diluent for 5 min
at 40 8C. Subsequently, they were incubated in
ELISA-positive murine anti-MHV serum (diluted 1
in 60 in M.O.M.e diluent) as the primary antibody
for 24 h at 4 8C. Substitution of PBS or mouse
serum (negative for any pathogen) for the primary
antibody served as a negative control. The sections
were then rinsed in PBS, incubated with M.O.M.e
biotinylated anti-mouse IgG reagent for 60 min
at 40 8C, rinsed in PBS, incubated in Vectastainw
ABC-AP reagent for 60 min at room temperature,
rinsed in PBS, and incubated with alkaline phos-
phatase substrate solution (Vectorw
Red; Vector
Laboratories) in 100 mM Tris HCl, pH 8.2–8.5, for
30 min at room temperature. Endogenous alkaline
phosphatase activity of tissues was inhibited
by adding one drop of levamisole (Vector
Laboratories) to 5 ml of Tris HCl buffer before
preparation of the substrate working solution. The
sections were rinsed in distilled water, counter-
stained with Mayer’s haematoxylin and examined
microscopically.
Lung sections from experiment 2 were pre-
treated as described in experiment 1. They were
then rinsed in PBS, incubated for 30 min at 40 8C in
diluted (1 in 20) normal horse serum (Vector
Laboratories), incubated with diluted (1 in 60)
ELISA-positive murine anti-M. pulmonis serum
(ELISA score:13.4–17.7) as primary antibody for
24 h at 4 8C, rinsed in PBS, and incubated with
diluted biotinylated horse anti-mouse IgG second-
ary antibody (1 in 100, rat-absorbed) for 60 min at
40 8C. The subsequent procedures were the same as
those in experiment 1.
Results
ELISA Monitoring Results
In the MHV test, 485 mouse serum samples were
examined. Of these, 465 were negative (score ,3),
three were positive (score 3–10), and 17 had a high
MHV score (.10). Seven of 17 samples with a high
score were excluded because of simultaneous
positivity for GDVII. Ten serum samples (ELISA
score: 11.2–14.4) were collected for further diag-
nosis of MHV infection in experiment 1.
In the M. pulmonis test, 268 rat serum samples
and 383 mouse serum samples were examined.
Only six rat samples were positive, and 380 mouse
samples were negative (score ,3). Three mouse
samples showed a high M. pulmonis score (.10),
but one of these was excluded because of simul-
taneous positivity for MHV. The remaining two
mouse serum samples (ELISA score: 13.4–17.7)
were used for the diagnosis of M. pulmonis infection
in Wistar rats in experiment 2. These Wistar rats
C.T. Liang et al.216

4. were confirmed as having M. pulmonis infection by
testing 13 rat serum samples; three rats (23%) had
an ELISA score of 6.07–17.22, and four (31%) had
typical pulmonary lesions.
Histopathology and Immunohistochemistry
Experiment 1. Gross examination of the four affected
male nude mice revealed that the liver was firm and
pale with multiple, white, depressed foci, 2 to 3 mm
in diameter. Microscopically, necrotic foci were
seen to be scattered throughout the hepatic
parenchyma, being well demarcated from the
adjacent normal tissues (Fig. 1). Multinucleated
syncytial giant cells with basophilic cytoplasmic
granules (20 mm in diameter) were often present.
Colonic and caecal mucosal ridges were attenuated
and shortened, and contained syncytial cells
(Fig. 2). Immunohistochemical results revealed
MHV antigen at the periphery of the necrotic foci
in the liver (Fig. 3) and within the caecal and
colonic multinucleated syncytial cells (Fig. 4),
spleen, and jejunal and gastric mucosa.
Experiment 2. Microscopical examination of two
affected Wistar rats showed extensive consolidation
of the lung, with variable degrees of hyperplasia
and metaplasia of bronchiolar epithelial cells, and
mononuclear cell infiltration into the bronchioles
and adjacent alveolar spaces. Loss of cilia and
flattening of epithelial cells in the bronchioles were
noted (Fig. 5). Aggregates of necrotic debris and
neutrophils were present in the bronchiolar
lumina. Peribronchial and perivascular cuffing
by lymphocytes, macrophages and plasma cells
was also observed. Sections of lung showed
immunohistochemical labelling for M. pulmonis
Fig. 1. MHV-infected mouse liver showing necrosis with multi-
nucleated syncytial giant cells (arrow) at the periphery
of the lesion. HE. Bar, 50 mm.
Fig. 2. Syncytial cells (arrowhead) in the surface epithelium of
the colon of a MHV-infected mouse. HE. Bar, 25 mm.
Fig. 3. MHV antigen within necrotic hepatocytes and syncytial
cells (arrowhead) immunolabelled with ELISA-positive
serum as primary antibody. ABC-AP with haematoxylin
counterstain. Bar, 25 mm.
Fig. 4. Colonic epithelial and syncytial cells (arrowheads)
immunolabelled for MHV with ELISA-positive sera.
ABC-AP with haematoxylin counterstain. Bar, 25 mm.
Diagnosis of Laboratory Animal Infections 217

5. antigen over the luminal surface of hyperplastic
bronchiolar epithelial cells (Fig. 6).
Discussion
Only 10 serum samples with a positive ELISA titre
for MHV alone and two with a positive titre for
M. pulmonis alone were used in this study. The
results indicated the applicability of the technique
to diagnosis in the absence of access to commer-
cially produced antibodies. There have been few
reports of the use of ELISA-positive murine sera as
primary antibody for the immunohistochemical
examination of rodent tissue. Polyclonal antiserum
has been used for the diagnosis of MHV and
M. pulmonis infection (Brownstein and Barthold,
1982; Brunnert et al., 1994; Liang et al., 1995). MHV
is the most common viral pathogen of mice, and
seropositivity for MHV had been reported in
19–83% of animals in mouse colonies (Kraft and
Meyer, 1986, 1990; Casebolt et al., 1988; National
Research Council, 1991). In Taiwan, MHV is highly
prevalent in mouse colonies, especially in immu-
nosuppressed mice (Liang et al., 1995). Due to the
inability of immunodeficient homozygous mice to
produce antibody, immunohistochemistry may
represent a useful replacement for serology in
diagnosing MHV infection. In this study, high-titre
serum from immunocompetent mice was used as
primary antibody. The findings regarding the
distribution of MHV antigen in experiment 1
accorded with those of previous reports (Weir
et al., 1987; Barthold et al., 1990), the antigen
being demonstrated in the intestine, liver, spleen
and stomach, consistent with multi-organ MHV
infection (Compton et al., 1993).
Serology and culture are widely used in the
diagnosis of M. pulmonis infection, but discrepan-
cies sometimes occur (Cassell et al., 1981). ELISA
has detected M. pulmonis infection in 8–78% of rat
colonies and 35–91% of mouse colonies, depend-
ing on whether conventional or barrier-maintained
facilities are used (Casebolt et al., 1988; Kraft and
Meyer, 1990). An advantage of ELISA is the low
incidence of non-specific or false positive reactions
as compared with haemagglutination inhibition
(HI) (Kraft and Meyer, 1986). Discrepant results
for M. pulmonis infection obtained by different
serological tests may be due to reactive substances
in the serum, such as lysozyme, antinuclear
antibodies, protease and bacterial products
(LaRegina et al., 1987). Culture of M. pulmonis
from tracheobronchial lavage fluid showed 89.6%
positivity in rats and 36.5% positivity in mice in
non-barrier-maintained facilities (Timenetsky and
DeLuca, 1998). For routine monitoring of
M. pulmonis, the preferred use of time-consuming
culture procedures as opposed to serological
testing is applicable only in acute or early infection.
One-third of infected animals do not yield
M. pulmonis in culture (Kraft et al., 1982). Culture
and histopathology may be misleading in evaluat-
ing a colony of rodents for mycoplasma infection,
particularly when the prevalence is low (Cox et al.,
1988).
M. pulmonis infection in the chronic stage is
readily detected histopathologically (Kraft et al.,
1982; Goto et al., 1994), but in some instances MHV
or mycoplasmal infection produces minimal or no
lesions. In such instances, immunohistochemistry
is valuable (Matthaei et al., 1998). In experiment 2,
labelling of M. pulmonis antigen was noted on
Fig. 5. Hyperplasia of bronchiolar epithelial cells of a rat
infected with M. pulmonis. HE. Bar, 25 mm.
Fig. 6. M. pulmonis antigen is clearly demonstrated on the
luminal surface of hyperplastic bronchiolar epithelial
cells. ABC-AP with haematoxylin counterstain. Bar,
25 mm.
C.T. Liang et al.218

6. the luminal surface of bronchiolar epithelial cells, a
site also affected by the cilia-associated respiratory
(CAR) bacillus (Matsushita et al., 1987). In a
previous study (Brunnert et al., 1994) the ABC
method failed to detect M. pulmonis in formalin-
fixed lung tissue but gave 27.4% (17/62) positive
results with ethanol-fixed lung tissue; this com-
pared with 96% (60/62) positive results given by
the polymerase chain reaction (PCR). However,
MHV antigens were demonstrated in formalin-
fixed, paraffin wax-embedded blocks for as long
as 2 years after preparation (Brownstein and
Barthold, 1982). Immunohistochemistry, com-
monly used to detect rodent pathogens, takes
much less time than that required for culturing
mycoplasmas. In the present study, however, the
incubation with primary antiserum was carried out
overnight at 4 8C (Miller and van der Maaten,
1989), rather than for 30 min at room temperature
or at 40 8C (Liu et al., 1997; Liang et al., 2000), the
purpose being to increase the immunolabelling.
PCR is more sensitive than immunohistochem-
istry or microbial isolation but requires a high
degree of technical expertise, and contamination
leads to false positivity (Brunnert et al., 1994). It
offers an advantage over serological testing, how-
ever, in situations in which an antibody response is
unlikely to be generated. In conclusion, the
method described, in which ELISA-positive serum
was used as primary antibody, may be useful in
diagnosing infection in immunodeficient animals
or when commercial immunohistochemical
reagents are unavailable (Matthaei et al., 1998).
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Received; May 6th; 2003
Accepted; April 7th; 2004
C.T. Liang et al.220